This invention relates to an assay wherein hydrogen peroxide is used as an assay reagent, and wherein hydrogen peroxide in a stable, dry hydrogen peroxide adduct form is used without inducing any adverse effects on the assay principle.
Typical assays wherein peroxide is used as an assay reagent are chemiluminescence assays. For example, in a chemiluminescence reaction known as peroxalate chemiluminescence, a high energy intermediate is generated from an oxalic acid derivative and hydrogen peroxide in the presence of a basic catalyst, and a fluorescent reagent is excited by the resulting energy to thereby induce luminescence. In the case of a luminol derivative, it is oxidized by hydrogen peroxide in the presence of Fe(CN).sub.6.sup.3-, microperoxidase, or peroxidase to result in the excited state of aminophthalic acid, and the luminescence occurs upon returning of the excited aminophthalic acid to its ground state. An acridinium derivative also undergoes luminescence with the addition of hydrogen peroxide. Such chemiluminescence reactions are widely used in various assays including those based on a specific binding reaction, and in such assays, the luminescence compound or the catalyst (such as enzyme) for the luminescence reaction is incorporated as a label. Hydrogen peroxide is also incorporated in such assay as a reagent indispensable for inducing the luminescence. The chemiluminescence reactions as described above exhibit a relatively high sensitivity, and therefore, these reactions are widely employed for the assays in various fields including clinical tests and environmental tests.
The assays wherein hydrogen peroxide is used as an assay reagent are not limited to the chemiluminescence assays. Peroxidase enzymes such as horseradish peroxidase (hereinafter referred to as HRPO) and microperoxidase are also used for the catalyst of color development reactions and electrochemical reactions. HRPO is used for the labeling enzyme in many assays including enzyme immunoassays, nucleic acid hybridization assays, and immunohistostaining assays wherein a tissue or cells are stained with a labeled antibody specific therefor, and in such assays, hydrogen peroxide, which is the substrate for the HRPO, is used as an assay reagent. Hydrogen peroxide is also used in occult blood test of feces wherein peroxidase activity of hemoglobin in feces is detected by color development reaction and hydrogen peroxide is added as the substrate of the enzymatic reaction. Other catalysts having peroxidase activity include ions and cheletes of a metal, porphyrin derivatives, catalyst antibodies, and the like, and these catalysts are also used in various assays wherein peroxide is used as an assay reagent as in the case of the enzyme catalysts as described above.
Recently, MEDIA (mediator diffusion-controlled immunoassay) has been disclosed as a specific binding assay wherein a signal substance generator such as an enzyme is used for the labeling agent to be developed through the matrix with the liquid sample; and the labeling agent is allowed to form a distribution of a particular profile in terms of the distance of the labeling agent from the detection port by the specific binding reaction between the analyte in the liquid sample and the specific binding substance; and the current corresponding to the analyte concentration of the liquid sample which is controlled by diffusion of the signal substance such as an electron mediator generated from the labeling agent is measured to thereby determine the concentration of the analyte. (See Japanese Patent Application Laid-Open No. 5(1993)-264552(EP 0 525 723 A2) and Japanese Patent Application Laid-Open No. 7(1995)-234201.) In MEDIA, HRPO is again used for the labeling agent in view of its high sensitivity as an oxidoreductase, and hydrogen peroxide is used in combination with the HRPO as the substrate for the label enzyme HRPO.
In the wide variety of assays as described above, the principle that a reaction specific for the analyte is detected by utilizing hydrogen peroxide has been adopted as would be appreciated from the foregoing description. A process capable of providing the hydrogen peroxide in stable, dry state without inducing any adverse effect on the assay principle is urgently required both for the convenience of the assay procedure and for the stability of the assay reagent.
Conventional means of retaining the hydrogen peroxide in dry state include dried urea-hydrogen peroxide adduct (Hyperole) and dried sodium carbonate-hydrogen peroxide adduct. These adducts are used in other fields as a bleach, a cleaner, a hair dye, and the like. The hydrogen peroxide adducts with urea and sodium carbonate are highly moisture absorptive and unstable, and could not endure long-term storage. Furthermore, urea is a compound which is well known as a denaturing agent of proteins, and accordingly, suffered from the defects that its use resulted in adverse effects when used in the assays as described above wherein proteins were involved as the analyte or assay reagents. Sodium carbonate also suffered from the detect of the high pH after its dissolution, and such defect inhibited its use in the assay purposes.
The MEDIA (mediator diffusion-controlled immunoassay) as described above is a specific binding assay provided with the feature of a dry chemistry assay wherein no washing operation is required and the assay can be conducted by merely adding the liquid sample to the assay device. In the MEDIA device described in Japanese Patent Application Laid-Open No. 7(1995)-234201, hydrogen peroxide in dry state was incorporated in the assay device in the form of urea-hydrogen peroxide adduct. In this device, however, the urea-hydrogen peroxide adduct in dry state had to be located at the downstream end of the assay device to prevent the adverse influence of urea on the antigen-antibody reaction and the enzyme reaction. Due to such remote location of the urea from the reaction site, a considerable time period was required for the establishment of stable response and quick measurement could not be realized in spite of the various measures taken for the promotion of natural diffusion of the hydrogen peroxide back to the reaction site. In addition, despite such remote location of the urea-hydrogen peroxide adduct from the reaction site, the assay device still suffered from inhibitory effects on the specific reaction of the urea that reversely diffused along the flow path after a while.